Advances in Cysteine Protease B Inhibitors for Leishmaniasis Treatment.

The expression and release of cysteine proteases by Leishmania spp. and their virulence factors significantly influence the modulation of host immune responses and metabolism, rendering cysteine proteases intriguing targets for drug development. This review article explores the substantial role of cysteine protease B (CPB) in medicinal chemistry from 2001 to 2024, particularly concerning combatting Leishmania parasites. We delve into contemporary advancements and potential prospects associated with targeting cysteine proteases for therapeutic interventions against leishmaniasis, emphasizing drug discovery in this context. Computational analysis using the pkCSM tool assessed the physicochemical properties of compounds, providing valuable insights into their molecular characteristics and drug-like potential, enriching our understanding of the pharmacological profiles, and aiding rational inhibitor design. Our investigation highlights that while nonpeptidic compounds constitute the majority (69.2%, 36 compounds) of the dataset, peptidomimetic- based derivatives (30.8%, 16 compounds) also hold promise in medicinal chemistry. Evaluating the most promising compounds based on dissociation constant (Ki) and half maximal inhibitory concentration (IC50) values revealed notable potency, with 41.7% and 80.0% of nonpeptidic compounds exhibiting values < 1 µM, respectively. On the other hand, all peptidic compounds evaluated for Ki (43.8%) and IC50 (31.3%) obtained values < 1 µM, respectively. Further analysis identified specific compounds within both categories (nonpeptidic: 1, 2, and 4; peptidic: 48-52) as particularly promising, warranting deeper investigation into their structure-activity relationships. These findings underscore the diverse landscape of inhibitors in medicinal chemistry and highlight the potential of both nonpeptidic and peptide-based compounds as valuable assets in therapeutic development against leishmaniasis.

Design and Synthesis of Benzimidazole Carboxamide Cysteine Protease Inhibitors as Promising Anti-leishmanial Agents.

INTRODUCTION: More than 20 protozoan species of Leishmania are responsible for causing Leishmaniasis, an infection spread by blood-feeding phlebotomine sandflies. A narrow pool of drugs is currently available rendering the current drug stratagem to treat this infection . Development of novel, less toxic, and more effective regimens is thus a need of the hour. Design and synthesis of benzo[d]imidazole carboxamides as agents to combat Leishmaniasis are also required. METHODS: 14 benzo[d]imidazole carboxamides were synthesized and gauged against L. donovani promastigotes and intramacrophage amastigote forms. All of the tested compounds exhibited significant anti-promastigote properties with IC50 well below 10 uM. Compounds 4a, 4b, and 4d, showing the highest anti-parasitic activity against promastigote forms (IC50 0.91- 1.33 µM), were also found to be associated with better anti-leishmanial potential (IC50 0.78- 1.67 µM) against the intramacrophage amastigotes comparable to Amphotericin-B (0.13 µM), a drug used for Leishmaniasis. Compound (4a), namely N-(2-(trifluoromethyl)-1Hbenzo[ d]imidazol-5-yl)benzo[d][1,3]-5-carboxamide-dioxole, was found to be most potent against L. donovani amastigotes among all the tested compounds, and demonstrated better antileishmanial properties (IC50 0.78 µM) when compared to the standard. Compound 4a was also assessed for its toxicity profile against THP-1 human monocytic cells. To establish the molecular target(s) in silico, molecular docking studies were performed against cysteine protease, a putative virulence factor of Leishmania parasites, and nucleoside diphosphate kinase, an enzyme with a critical role in nucleotide recycling, also associated with resistance in Leishmania strains. Compound 4a showed better binding affinity than the standard to these targets; furthermore, the molecular dynamic simulation studies further affirmed the stability of compound 4a, within the active site of the targets. In vitro, cysteine protease inhibitory activity (IC50 8.53 µM) using Bz-Arg-AMC hydrochloride fluorogenic peptide substrate established the promising potential of 4a as a cysteine protease inhibitor. RESULT: Computational ADMET analysis indicated appropriate pharmacokinetic profile and physicochemical characteristics for all members of the synthesized library. CONCLUSION: Both in vitro and in silico studies indicate that the synthesized imidazole carboxamides can act as potent hits and that N-(2-(trifluoromethyl)-1H-benzo[d]imidazol-5- yl)benzo[d][1,3]-5-carboxamide-dioxole 4a can be an effective hit molecule which can be further developed into potent lead molecule (s) to fight Leishmania donovani.

SARS-CoV-2 Genotyping Highlights the Challenges in Spike Protein Drift Independent of Other Essential Proteins.

As of 2024, SARS-CoV-2 continues to propagate and drift as an endemic virus, impacting healthcare for years. The largest sequencing initiative for any species was initiated to combat the virus, tracking changes over time at a full virus base-pair resolution. The SARS-CoV-2 sequencing represents a unique opportunity to understand selective pressures and viral evolution but requires cross-disciplinary approaches from epidemiology to functional protein biology. Within this work, we integrate a two-year genotyping window with structural biology to explore the selective pressures of SARS-CoV-2 on protein insights. Although genotype and the Spike (Surface Glycoprotein) protein continue to drift, most SARS-CoV-2 proteins have had few amino acid alterations. Within Spike, the high drift rate of amino acids involved in antibody evasion also corresponds to changes within the ACE2 binding pocket that have undergone multiple changes that maintain functional binding. The genotyping suggests selective pressure for receptor specificity that could also confer changes in viral risk. Mapping of amino acid changes to the structures of the SARS-CoV-2 co-transcriptional complex (nsp7-nsp14), nsp3 (papain-like protease), and nsp5 (cysteine protease) proteins suggest they remain critical factors for drug development that will be sustainable, unlike those strategies targeting Spike.

Cowpea lipid transfer protein 1 regulates plant defense by inhibiting the cysteine protease of cowpea mosaic virus.

Many virus genomes encode proteases that facilitate infection. The molecular mechanism of plant recognition of viral proteases is largely unexplored. Using the system of Vigna unguiculata and cowpea mosaic virus (CPMV), we identified a cowpea lipid transfer protein (LTP1) which interacts with CPMV-encoded 24KPro, a cysteine protease, but not with the enzymatically inactive mutant 24KPro(C166A). Biochemical assays showed that LTP1 inhibited 24KPro proteolytic cleavage of the coat protein precursor large coat protein-small coat protein. Transient overexpression of LTP1 in cowpea reduced CPMV infection, whereas RNA interference-mediated LTP1 silencing increased CPMV accumulation in cowpea. LTP1 is mainly localized in the apoplast of uninfected plant cells, and after CPMV infection, most of the LTP1 is relocated to intracellular compartments, including chloroplast. Moreover, in stable LTP1-transgenic Nicotiana benthamiana plants, LTP1 repressed soybean mosaic virus (SMV) nuclear inclusion a protease activity, and accumulation of SMV was significantly reduced. We propose that cowpea LTP1 suppresses CPMV and SMV accumulation by directly inhibiting viral cysteine protease activity.

MicroED structure of the C11 cysteine protease clostripain.

Clostripain secreted from Clostridium histolyticum is the founding member of the C11 family of Clan CD cysteine peptidases, which is an important group of peptidases secreted by numerous bacteria. Clostripain is an arginine-specific endopeptidase. Because of its efficacy as a cysteine peptidase, it is widely used in laboratory settings. Despite its importance the structure of clostripain remains unsolved. Here we describe the first structure of an active form of C. histolyticum clostripain determined at 2.5 Å resolution using microcrystal electron diffraction (MicroED). The structure was determined from a single nanocrystal after focused ion beam milling. The structure of clostripain shows a typical Clan CD α/ß/α sandwich architecture and the Cys231/His176 catalytic dyad in the active site. It has a large electronegative substrate binding pocket showing its ability to accommodate large and diverse substrates. A loop in the heavy chain formed between residues 452 and 457 is potentially important for substrate binding. In conclusion, this result demonstrates the importance of MicroED to determine the unknown structure of macromolecules such as clostripain, which can be further used as a platform to study substrate binding and design of potential inhibitors against this class of peptidases.

Identification of Dihydropyrazolo[1,5-a]pyrazin-4(5H)-ones as Cyclic Products of ß-Amidomethyl Vinyl Sulfone Alphavirus Cysteine Protease Inhibitors.

Optimized syntheses of (E)-5-(2-ethoxyphenyl)-N-(3-(methylsulfonyl)allyl)-1H-pyrazole-3-carboxamide (RA-0002034, 1), a promising antiviral covalent cysteine protease inhibitor lead, were developed. The syntheses avoid the contamination of 1 with the inactive cyclic dihydropyrazolo[1,5-a]pyrazin-4(5H)-one 2, which is formed by the intramolecular aza-Michael reaction of the vinyl sulfone warhead under basic conditions and slowly at pH 7.4 in phosphate buffer. The pure cysteine protease inhibitor 1 could be synthesized using either modified amide coupling conditions or through the introduction of a MOM-protecting group and was stable as a TFA or HCl salt. Although acyclic 1 demonstrated poor pharmacokinetics with high in vivo clearance in mice, inactive cyclic 2 showed improved plasma exposure. The potential use of cyclic dihydropyrazolo[1,5-a]pyrazin-4(5H)-ones as prodrugs for the acyclic ß-amidomethyl vinyl sulfone warhead was demonstrated by GSH capture experiments with an analog of 2.

Comparing the Yield of Recombinant Human Factor VII Protein Expressed by the rDNA-Promoter with the CMV-Promoter in Iranian Lizard Leishmania.

Background: Iranian Lizard Leishmania (I.L.L) is a nonpathogenic Leishmania strain. Due to its advantages, several recombinant proteins have been produced in this host. However, I.L.L shows a lower yield of recombinant protein expression compared to other commercial hosts. Considering the role of protease enzymes in protein digestion, we selected cysteine protease B (CPB) to investigate its impact on recombinant protein yield in I.L.L. Methods: we generated gene knockouts by utilizing homologous recombination (HR) and CRISPR methods. To assess the efficacy of the designed construct, we compared the yield of recombinant human factor VII (rhFVII) production between cells transfected with the pLEXSY-hyg2-FVII vector and the CMV-promoter-based construct (pF7cmvneo). Results: The knockout of a single CPB gene allele through the HR method or the complete knockout of all alleles through the CRISPR method led to cell death. This outcome suggests that even the deletion of a single CPB gene allele diminishes the protein to a level insufficient for the survival of I.L.L, indicating a critical dependency on the presence of this protein for the organism's viability. rhFVII exhibited a greater expression yield with the pLEXSY construct compared to the pF7cmvneo construct in I.L.L. The lower expression rate of pF7cmvneo may be influenced by epigenetic factors related to the CPC gene or the RNA polymerase used for the expression of that promoter. Conclusion: Therefore, considering alternative integration targets for CMV-promoter-based constructs and incorporating UTR sequences of I.L.L high-expression proteins in the vector may enhance recombinant protein expression rates.

Natural Compounds as Protease Inhibitors in Therapeutic Focus on Cancer Therapy.

Proteases are implicated in every hallmark of cancer and have complicated functions. For cancer cells to survive and thrive, the process of controlling intracellular proteins to keep the balance of the cell proteome is essential. Numerous natural compounds have been used as ligands/ small molecules to target various proteases that are found in the lysosomes, mitochondria, cytoplasm, and extracellular matrix, as possible anticancer therapeutics. Promising protease modulators have been developed for new drug discovery technology through recent breakthroughs in structural and chemical biology. The protein structure, function of significant tumor-related proteases, and their natural compound inhibitors have been briefly included in this study. This review highlights the most current frontiers and future perspectives for novel therapeutic approaches associated with the list of anticancer natural compounds targeting protease and the mode and mechanism of proteinase-mediated molecular pathways in cancer.

Cysteine protease cathepsin B promotes lysosome integrity to extend the lifespan of alternative day fasting worms.

Alternative day fasting (ADF) has been shown to enhance the lifespan of animals. However, human trials evaluating the efficacy of ADF have only recently emerged, presenting challenges due to the extreme nature of this dietary regimen. To better understand the effects of ADF, we investigated its impact using Caenorhabditis elegans as a model organism. Our findings reveal that ADF extends the lifespan of worms nourished on animal-based protein source, while those fed with plant-based protein as the primary protein source do not experience such benefits. Remarkably, initiating ADF during midlife is sufficient to prolong lifespan, whereas implementation during youth results in developmental damage, and in older age, fails to provide additional extension effects. Furthermore, we discovered that midlife ADF up-regulates the expression of two cysteine protease cathepsin B genes, cpr-2 and cpr-5, which preserve lysosomal integrity and enhance its function in digesting aggregated proteins, as well as enhancing lipid metabolism and ameliorating neurodegenerative disease markers and phenomena during aging. This suggests that midlife ADF has long lasting anti-aging effects and may delay the onset of related diseases, specifically in animals consuming animal-based protein source. These findings offer valuable insights into the effects of ADF and provide guidance for future research and potential applications in individuals.

Exploring the extraction, functional properties, and industrial applications of papain from Carica papaya.

Papain a protease enzyme naturally present in the Carica papaya has gained significant interest across several industries due to its unique properties and versatility. The unique structure of papain imparts the functionality that assists in elucidating how papain enzyme works and making it beneficial for a variety of purposes. This review highlights recent advancements in papain extraction techniques to enhance production efficiency to meet market demand. The extraction of papain from the Carica papaya plant offers various advantages such as cost-effectiveness, biodegradability, safety, and the ability to withstand a wide range of pH and temperature conditions. Key findings reveal that non-conventional papain extraction techniques offer significant advantages in terms of efficiency, product quality, and environmental sustainability. Furthermore, papain treatment enhances the value of final products due to its anti-bacterial, anti-oxidant, and anti-obesity properties. The ability of papain to hydrolyze a wide range of proteins across various conditions makes it a suitable protease enzyme. While the study emphasizes the advantages of papain, the study also acknowledges limitations such as the continuous research and development to optimize extraction processes which will help unlock papain's potential and meet the growing demand. © 2024 Society of Chemical Industry.

Novel selective proline-based peptidomimetics for human cathepsin K inhibition.

Human cathepsin K (CatK) stands out as a promising target for the treatment of osteoporosis, considering its role in degrading the bone matrix. Given the small and shallow S2 subsite of CatK and considering its preference for proline or hydroxyproline, we now propose the rigidification of the leucine fragment found at the P2 position in a dipeptidyl-based inhibitor, generating rigid proline-based analogs. Accordingly, with these new proline-based peptidomimetics inhibitors, we selectively inhibited CatK against other human cathepsins (B, L and S). Among these new ligands, the most active one exhibited a high affinity (pKi = 7.3 - 50.1 nM) for CatK and no inhibition over the other cathepsins. This specific inhibitor harbors two novel substituents never employed in other CatK inhibitors: the trifluoromethylpyrazole and the 4-methylproline at P3 and P2 positions. These results broaden and advance the path toward new potent and selective inhibitors for CatK.

Correlation Between Blood Levels of Cysteine Protease Caspase-1 (Casp1) and Pain Scores (NRS) Post-surgery: A Prospective Randomized Study of Patients With Cholelithiasis.

BACKGROUND/AIM: Cysteine protease caspase-1 (Casp1) plays a crucial role in the conversion of pro-cytokines to active cytokines (CYTs). The purpose of this work was to determine Casp1 blood levels in a cohort of 114 cholecystectomy patients and assess their association with other CYTs and numeric rating scale (NRS) pain scores, postoperatively. PATIENTS AND METHODS: Blood levels of Casp1 and seven CYTs (IL-18, IL-18BP, IL-1ra, IL-6, IL-10, IL-1ß, and IL-8) were measured at three time points; before operation, immediately after operation, and six hours after operation in 114 patients with cholelithiasis (Chole). RESULTS: Casp1 blood levels correlated with NRS pain scores at 24 h following surgery (p=0.016). In addition, Casp1 blood levels correlated significantly to IL-18 blood levels (p<0.001). CONCLUSION: This is the first report to evaluate Casp1 blood levels in Chole patients in correlation with other CYTs. The findings confirm a significant correlation between Casp1 blood levels and NRS pain scores. Moreover, this study provides initial evidence suggesting that inhibition of the activity of Casp1 may reduce postsurgical acute phase immune response possibly through the Casp1/pro-Il-18 pathway.

Halogenated Secondary Metabolites from Higher Plants: Potent Drug Candidates for Chikungunya Using in silico Approaches.

Chikungunya virus (CHIKV) causes a debilitating fever and joint pain, with no specific antiviral treatment available. Halogenated secondary metabolites from plants are a promising new class of drug candidates against chikungunya, with unique properties that make them effective against the virus. Plants produce these compounds to defend themselves against pests and pathogens, and they are effective against a wide range of viruses, including chikungunya. This study investigated the interactions of halogenated secondary metabolites with nsP2pro, a therapeutic target for CHIKV. A library of sixty-six halogenated plant metabolites screened previously for ADME properties was used. Metabolites without violation of Lipinski's rule were docked with nsP2pro using AutoDock Vina. To find the stability of the pipoxide chlorohydrin-nsP2pro complex, the GROMACS suite was used for MD simulation. The binding free energy of the ligand-protein complex was computed using MMPBSA. Molecular docking studies revealed that halogenated metabolites interact with nsP2pro, suggesting they are possible inhibitors. Pipoxide chlorohydrin showed the greatest affinity to the target. This was further confirmed by the MD simulations, surface accessible area, and MMPBSA studies. Pipoxide chlorohydrin, a halogenated metabolite, was the most potent against nsP2pro in the survey.

Plasmodium falciparum cysteine protease Falcipain 3: A potential enzyme for proteolytic processing of histone acetyltransferase PfGCN5.

In spite of 150 years of studying malaria, the unique features of the malarial parasite, Plasmodium, still perplex researchers. One of the methods by which the parasite manages its gene expression is epigenetic regulation, the champion of which is PfGCN5, an essential enzyme responsible for acetylating histone proteins. PfGCN5 is a ∼170 kDa chromatin-remodeling enzyme that harbors the conserved bromodomain and acetyltransferase domain situated in its C-terminus domain. Although the PfGCN5 proteolytic processing is essential for its activity, the specific protease involved in this process still remains elusive. Identification of PfGCN5 interacting proteins through immunoprecipitation (IP) followed by LC-tandem mass spectrometry analysis revealed the presence of food vacuolar proteins, such as the cysteine protease Falcipain 3 (FP3), in addition to the typical members of the PfGCN5 complex. The direct interaction between FP3 and PfGCN5 was further validated by in vitro pull-down assay as well as IP assay. Subsequently, use of cysteine protease inhibitor E64d led to the inhibition of protease-specific processing of PfGCN5 with concomitant enrichment and co-localization of PfGCN5 and FP3 around the food vacuole as evidenced by confocal microscopy as well as electron microscopy. Remarkably, the proteolytic cleavage of the nuclear protein PfGCN5 by food vacuolar protease FP3 is exceptional and atypical in eukaryotic organisms. Targeting the proteolytic processing of GCN5 and the associated protease FP3 could provide a novel approach for drug development aimed at addressing the growing resistance of parasites to current antimalarial drugs.

Schistosoma japonicum cystatin alleviates paraquat poisoning caused acute lung injury in mice through activating regulatory macrophages.

BACKGROUND: Paraquat (PQ) is a widely used herbicide that poisons human by accident or intentional ingestion. PQ poisoning causes systemic inflammatory response syndrome (SIRS) resulting in acute lung injury (ALI) with an extremely high mortality rate. Blood trematode Schistosoma japonicum-produced cystatin (Sj-Cys) is a strong immunomodulatory protein that has been experimentally used to treat inflammation related diseases. In this study, Sj-Cys recombinant protein (rSj-Cys) was used to treat PQ-induced lung injury and the immunological mechanism underlying the therapeutic effect was investigated. METHODS: PQ-induced acute lung injury mouse model was established by intraperitoneally injection of 20 mg/kg of paraquat. The poisoned mice were treated with rSj-Cys and the survival rate was observed up to 7 days compared with the group without treatment. The pathological changes of PQ-induced lung injury were observed by examining the histochemical sections of affected lung tissue and the wet to dry ratio of lung as a parameter for inflammation and edema. The levels of the inflammation related cytokines IL-6 and TNF-α and regulatory cytokines IL-10 and TGF-ß were measured in sera and in affected lung tissue using ELISA and their mRNA levels in lung tissue using RT-PCR. The macrophages expressing iNOS were determined as M1 and those expressing Arg-1 as M2 macrophages. The effect of rSj-Cys on the transformation of inflammatory M1 to regulatory M2 macrophages was measured in affected lung tissue in vivo (EKISA and RT-PCR) and in MH-S cell line in vitro (flow cytometry). The expression levels of TLR2 and MyD88 in affected lung tissue were also measured to determine their role in the therapy of rSj-Cys on PQ-induced lung injury. RESULT: We identified that treatment with rSj-Cys significantly improved the survival rate of mice with PQ-induced lung injury from 30 % (untreated) to 80 %, reduced the pathological damage of poisoning lung tissue, associated with significantly reduced levels of proinflammatory cytokines (IL-6 from 1490 to 590 pg/ml, TNF-α from 260 to 150 pg/ml) and increased regulatory cytokines (IL-10 from360 to 550 pg/ml, and TGF-ß from 220 to 410 pg/ml) in both sera (proteins) and affected lung tissue (proteins and mRNAs). The polarization of macrophages from M1to M2 type was found to be involved in the therapeutic effect of rSj-Cys on the PQ-induced acute lung injury, possibly through inhibiting TLR2/MyD88 signaling pathway. CONCLUSIONS: Our study demonstrated the therapeutic effect of rSj-Cys on PQ poisoning caused acute lung injury by inducing M2 macrophage polarization through inhibiting TLR2/MyD88 signaling pathway. The finding in this study provides an alternative approach for the treatment of PQ poisoning and other inflammatory diseases.

Extracellular proteolysis in cancer: Proteases, substrates, and mechanisms in tumor progression and metastasis.

A vast ensemble of extracellular proteins influences the development and progression of cancer, shaped and reshaped by a complex network of extracellular proteases. These proteases, belonging to the distinct classes of metalloproteases, serine proteases, cysteine proteases, and aspartic proteases, play a critical role in cancer. They often become dysregulated in cancer, with increases in pathological protease activity frequently driven by the loss of normal latency controls, diminished regulation by endogenous protease inhibitors, and changes in localization. Dysregulated proteases accelerate tumor progression and metastasis by degrading protein barriers within the extracellular matrix (ECM), stimulating tumor growth, reactivating dormant tumor cells, facilitating tumor cell escape from immune surveillance, and shifting stromal cells toward cancer-promoting behaviors through the precise proteolysis of specific substrates to alter their functions. These crucial substrates include ECM proteins and proteoglycans, soluble proteins secreted by tumor and stromal cells, and extracellular domains of cell surface proteins, including membrane receptors and adhesion proteins. The complexity of the extracellular protease web presents a significant challenge to untangle. Nevertheless, technological strides in proteomics, chemical biology, and the development of new probes and reagents are enabling progress and advancing our understanding of the pivotal importance of extracellular proteolysis in cancer.

Nature-Inspired Gallinamides Are Potent Antischistosomal Agents: Inhibition of the Cathepsin B1 Protease Target and Binding Mode Analysis.

Schistosomiasis, caused by a parasitic blood fluke of the genus Schistosoma, is a global health problem for which new chemotherapeutic options are needed. We explored the scaffold of gallinamide A, a natural peptidic metabolite of marine cyanobacteria that has previously been shown to inhibit cathepsin L-type proteases. We screened a library of 19 synthetic gallinamide A analogs and identified nanomolar inhibitors of the cathepsin B-type protease SmCB1, which is a drug target for the treatment of schistosomiasis mansoni. Against cultured S. mansoni schistosomula and adult worms, many of the gallinamides generated a range of deleterious phenotypic responses. Imaging with a fluorescent-activity-based probe derived from gallinamide A demonstrated that SmCB1 is the primary target for gallinamides in the parasite. Furthermore, we solved the high-resolution crystal structures of SmCB1 in complex with gallinamide A and its two analogs and describe the acrylamide covalent warhead and binding mode in the active site. Quantum chemical calculations evaluated the contribution of individual positions in the peptidomimetic scaffold to the inhibition of the target and demonstrated the importance of the P1' and P2 positions. Our study introduces gallinamides as a powerful chemotype that can be exploited for the development of novel antischistosomal chemotherapeutics.

Critical assessment of popular biomolecular force fields for molecular dynamics simulations of folding and enzymatic activity of main protease of coronavirus SARS-CoV-2.

The main cysteine protease (Mpro) of coronavirus SARS-CoV-2 has become a promising target for computational development in anti-COVID-19 treatments. Here, we benchmarked the performance of six biomolecular molecular dynamics (MD) force fields (OPLS-AA, CHARMM27, CHARMM36, AMBER03, AMBER14SB and GROMOS G54A7) and three water models (TIP3P, TIP4P and SPC) for reproducing the native fold and the enzymatic activity of Mpro as monomeric and dimeric units. The MD sampling up to 1 µs suggested that the proper choice of the force fields and water models plays an essential role in reproducing the tertiary structure and the inter-residue distance between the catalytic dyad His41-Cys145. We found that while most benchmarked all-atom force fields reproduce well the native fold of Mpro, the CHARMM27/TIP3P and OPLS-AA/TIP4P setups revealed a good performance in reproducing the structure of the catalytic domain. In addition, these FF setups were also well-adopted for MD sampling of Mpro at the physiologic conditions by mimicking the presence of 100 mM NaCl and the elevated temperature of 310 K. Finally, both FFs were also performed well in reproducing the native fold of Mpro in a dimeric form. Therefore, comparing the preservation of the native fold of Mpro and the stability of its catalytic site architecture, our MD benchmarking suggests that the OPLS-AA/TIP4P and CHARMM27/TIP3P MD setups at the physiologic conditions may be well-suited for rapid in silico screening and developing broad-spectrum anti-coronaviral therapeutic agents.

Use of E-64 cysteine protease inhibitor for the recombinant protein production in Tetrahymena thermophila.

Tetrahymena thermophila is an alternative organism for recombinant protein production. However, the production efficiency in T. thermophila is quite low mainly due to the rich cysteine proteases. In this study, we studied whether supplementation of the E-64 inhibitor to T. thermophila cultures increases the recombinant protein production efficiency without any toxic side effects. Our study showed that supplementation of E-64 had no lethal effects on T. thermophila cells in flask culture at 30 °C and 38 °C. In vitro protease activity analysis using secretome as protease enzyme source from E-64-supplemented cell cultures showed a reduced protein substrate degradation using bovine serum albumin, rituximab, and milk lactoglobulin proteins. E-64 also prevented proteolysis of the recombinantly produced and secreted TtmCherry2-sfGFP fusion protein at some level. This reduced inhibitory effect of E-64 could be due to genetic compensation of the inhibited proteases. As a result, the 5 µM concentration of E-64 was found to be a non-toxic protease inhibitory supplement to improve extracellular recombinant protein production efficiency in T. thermophila. This study suggests that the use of E-64 may increase the efficiency of extracellular recombinant protein production by continuously reducing extracellular cysteine protease activity during cultivation.

Mechanistic insights into CrCEP1: A dual-function cysteine protease with endo- and transpeptidase activity.

Proteases, essential regulators of plant stress responses, remain enigmatic in their precise functional roles. By employing activity-based probes for real-time monitoring, this study aimed to delve into protease activities in Chlamydomonas reinhardtii exposed to oxidative stress induced by hydrogen peroxide. However, our work revealed that the activity-based probes strongly labelled three non-proteolytic proteins-PsbO, PsbP, and PsbQ-integral components of photosystem II's oxygen-evolving complex. Subsequent biochemical assays and mass spectrometry experiments revealed the involvement of CrCEP1, a previously uncharacterized papain-like cysteine protease, as the catalyst of this labelling reaction. Further experiments with recombinant CrCEP1 and PsbO proteins replicated the reaction in vitro. Our data unveiled that endopeptidase CrCEP1 also has transpeptidase activity, ligating probes and peptides to the N-termini of Psb proteins, thereby expanding the repertoire of its enzymatic activities. The hitherto unknown transpeptidase activity of CrCEP1, working in conjunction with its proteolytic activity, unveils putative complex and versatile roles for proteases in cellular processes during stress responses.